Microscopy Plate Well 1B Cells | Well 2HEK 293 Permeabilized (Unstained) | Well 3 HEK293 Permeabilized | Well 4 HEK293 + Dummy (500ng) + hEF1a:mKate (500ng) Permeabilized | Well 5HEK293 + hEF1a:mKate (500ng) + hEF1a:PirB (500ng) Permeabilized | Well 6 | Well 7B Cells | Well 8HEK 293 Permeabilized (Unstained) | Well 9 HEK293 Permeabilized | Well 10 HEK293 + Dummy (500ng) + hEF1a:mKate (500ng) Permeabilized | Well 11HEK293 + hEF1a:mKate (500ng) + hEF1a:PirB (500ng) Permeabilized | Well 12 | Well 13B Cells | Well 14HEK 293 Non-Permeabilized (Unstained) | Well 15 HEK293 Non-Permeabilized | Well 16 HEK293 + Dummy (500ng) + hEF1a:mKate (500ng) Non- Permeabilized | Well 17HEK293 + hEF1a:mKate (500ng) + hEF1a:PirB (500ng) Non-Permeabilized | Well 18 | Well 19B Cells | Well 20HEK 293 Non-Permeabilized (Unstained) | Well 21 HEK293 Non-Permeabilized | Well 22 HEK293 + Dummy (500ng) + hEF1a:mKate (500ng) Non- Permeabilized | Well 23HEK293 + hEF1a:mKate (500ng) + hEF1a:PirB (500ng) Non-Permeabilized | Well 24 |
| Cytometry Plate Well 1B Cells | Well 2HEK 293 | Well 3 HEK293 | Well 4 HEK293 + Dummy (500ng) + hEF1a:mKate (500ng) | Well 5HEK293 + hEF1a:mKate (500ng) + hEF1a:PirB (500ng) | Well 6 | Well 7B Cells | Well 8HEK 293 | Well 9 HEK293 | Well 10 HEK293 + Dummy (500ng) + hEF1a:mKate (500ng) | Well 11HEK293 + hEF1a:mKate (500ng) + hEF1a:PirB (500ng) | Well 12
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Analysis:
Cytometry..
LilrB2 and PirB Trial 1: The plots for the mkate only and mkate + receptor cell populations are very similar. This is most probably due to the bleed through of the red channel into the yellow channel (the 'sensitivity' of the PMTs for the yellow channel was increased because the yellow fluorophore was not very bright).
Next steps:
- We are going to transfect the receptors without using a transfection marker (Trial 2).
Microscopy..
LilrB2 and PirB Trial 1: For both the receptors, there doesn't seem to be any clear localization of the receptors to the membrane. From the punctate appearance of the cells under the confocal, they seem to be significant localization of the receptors in some subcellular cytoplasmic structure. They may be making it to the membrane; however we are unable to discern that just from the images. Interestingly, this cytoplasmic localization seems to be apparent in the non-permeabilized cells which may mean that the fixing protocol is causing some kind of permeabilization in the cells. It may be of significance to note that in the LilrB2 paper (the one we are trying to replicate), they expressed the receptors under CMV (not hEF1a). Depending on the relative strengths of the promoters, the differing levels of expression (probably higher expression through hEF1a) might be causing the staining pattern that we are seeing.
Next steps:
- Perhaps staining live cells (to skip the fixation step) and hopefully conclude whether or not some of the receptor is making it to the membrane. (We are also going to do the beta amyloid binding experiment despite being unsure whether or not the receptor is making it to the membrane in hopes that it does bind and we can conclude that it is localizing.)
- Once we get the receptor-fluorescent protein fusions out of cloning, we can express them and look for localization. This will tell us whether or not the staining pattern that we're seeing is due to the protein expression or the staining.
- Clone the receptors into a different promoter: CMV or a TRE to determine if high expression levels are the problem. Having inducible expression of the receptors may be helpful later on in determining what the optimal level of receptor expression for the system.
